The present invention relates generally to methods for modulating OB-cadherin-mediated processes, and more particularly to the use of modulating agents comprising OB-cadherin cell adhesion recognition sequences, or antibodies that specifically recognize such sequences, for inhibiting or enhancing functions such as cell adhesion, tumor cell invasion and metastasis.
Cadherins are a rapidly expanding family of calcium-dependent cell adhesion molecules (CAMs) (for review, see Munro et al., In: Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp. 17-34, RG Landes Co., Austin, Tex., 1996). There are many different types of cadherins (abbreviated CADs). The most extensively studied group of CADs is known as the classical, or type I, CADs. These CADs are integral membrane glycoproteins that generally promote cell adhesion through homophilic interactions (a CAD on the surface of one cell binds to an identical CAD on the surface of another cell), although CADs also appear to be capable of forming heterotypic complexes with one another under certain circumstances and with lower affinity. Cadherins have been shown to regulate epithelial, endothelial, neural and cancer cell adhesion, with different CADs expressed on different cell types. N (neural)xe2x80x94cadherin is predominantly expressed by neural cells, endothelial cells and a variety of cancer cell types. E (epithelial)xe2x80x94cadherin is predominantly expressed by epithelial cells. Other CADs are P (placental)xe2x80x94cadherin, which is found in human skin and R (retinal)xe2x80x94cadherin. A detailed discussion of the classical cadherins is provided in Munro S B et al., In: Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp. 17-34 (RG Landes Company, Austin, Tex., 1996).
The structures of the CADs are generally similar. As illustrated in FIG. 1A, CADs are composed of five extracellular domains (EC1-EC5), a single hydrophobic domain (TM) that transverses the plasma membrane (PM), and two cytoplasmic domains (CP1 and CP2). The calcium binding motifs DXNDN (SEQ ID NO:1), DXD and LDRE (SEQ ID NO:2) are interspersed throughout the extracellular domains. The first extracellular domain (EC1) contains the classical cadherin cell adhesion recognition (CAR) sequence, HAV (His-Ala-Val), along with flanking sequences on either side of the CAR sequence that may play a role in conferring specificity. Synthetic peptides containing the CAR sequence and antibodies directed against the CAR sequence have been shown to inhibit CAD-dependent processes (Munro et al., supra; Blaschuk et al., J. Mol. Biol. 211:679-82, 1990; Blaschuk et al., Develop. Biol. 139:227-29, 1990; Alexander et al., J. Cell. Physiol. 156:610-18, 1993).
A second group of CADs is known as the atypical, or type II CADs (Munro et al., supra). Although the structure of these CADs is similar to that of the type I CADs, they do not contain the CAR sequence, HAV (FIG. 1B). Furthermore, the functions of the type II CADs are poorly understood.
OB-cadherin, which is also known as cadherin-11, is an atypical CAD (Okazaki et al., J. Biol. Chem. 269:12092-98, 1994; Suzuki et al., Cell Regulation 2:261-70, 1991; Munro et al., supra). This CAD can promote cell adhesion through homophilic interactions. Recent studies have shown that OB-cadherin is not expressed by well-differentiated, poorly invasive cancer cells, whereas it is expressed by invasive cancer cells (Stephen W. Byers, Georgetown University, Washington DC, personal communication; Shimazui et al., Cancer Res. 56:3234-37, 1996; Shibata et al., Cancer Letters 99:147-53, 1996). OB-cadherin levels are also high in stromal cells and osteoblasts (Shibata et al., Cancer Letters 99:147-53, 1996; Simonneau et al., Cell Adhes. Commun. 3:115-30, 1995; Matsuyoshi and Imamura, Biochem. Biophys. Res. Commun. 23:355-58, 1997; Okazaki et al., J. Biol. Chem. 269:12092-98, 1994). Collectively, these observations have led to the hypothesis that OB-cadherin may mediate the interaction between malignant tumor cells and other cell types, such as stromal cells and osteoblasts, thus facilitating tumor cell invasion and metastasis.
OB-cadherin is expressed in certain specific cell types. In some invasive cancer cells, OB-cadherin is not only found at sites of cellxe2x80x94cell contact, but it is also found in lamellopodia-like projections which do not interact with other cells (Stephen W. Byers, Georgetown University, Washington DC, personal communication). These observations suggest that OB-cadherin may also play a role in modulating cell-substrate interactions. In adipocytes, OB-cadherin is the only known expressed cadherin. OB-cadherin is therefore likely to mediate adhesion between adipocytes, and it is likely to be an important regulator of adipogenesis. Another cell type that expresses OB-cadherin is the pericyte (also known as the peri-endothelial cell). Pericytes are contractile cells which are similar to smooth muscle cells. They encircle the endothelial cells of blood vessels. Pericytes are involved in maintaining the structural integrity of blood vessels (Hanahan, Science 277:48-50, 1997; Lindahl et al., Science 277:242-245, 1997). Loss of pericytes causes blood vessels to regress.
Notwithstanding these recent advances, OB-cadherin function remains poorly understood at the biological and molecular levels. Accordingly, there is a need in the art for identifying sequences involved in modulating OB-cadherin-dependent cell adhesion, and for the development of methods employing such sequences to inhibit cancer cell adhesion, invasion and metastasis. The present invention fulfills these needs and further provides other related advantages.
Briefly stated, this invention provides compositions and methods for modulating OB-cadherin-mediated processes, such as cancer cell adhesion, invasion, and metastasis. Within certain aspects, cell adhesion modulating agents capable of modulating OB-cadherin mediated cell adhesion are provided. Such modulating agents may comprise at least one of: (a) a native OB-cadherin CAR sequence; (b) an analogue of a native OB-cadherin CAR sequence that is capable of modulating OB-cadherin-mediated cell adhesion; (c) a non-peptide peptidomimetic of an OB-cadherin CAR sequence that is capable of modulating OB-cadherin-mediated cell adhesion; (d) an antibody, or antigen-binding fragment thereof, that specifically binds an OB-cadherin CAR sequence; and/or (e) a polynucleotide encoding a native OB-cadherin CAR sequence or analogue thereof that is capable of modulating OB-cadherin-mediated cell adhesion. Certain preferred modulating agents may comprise a peptide that comprises one or more of the following peptide sequences: DDK, IDDK (SEQ ID NO:32), DDKS (SEQ ID NO:33), VIDDK (SEQ ID NO:3), IDDKS (SEQ ID NO:4), VIDDKS (SEQ ID NO:34), DDKSG (SEQ ID NO:35), IDDKSG (SEQ ID NO:24), VIDDKSG (SEQ ID NO:36), FVIDDK (SEQ ID NO:37), FVIDDKS (SEQ ID NO:38), FVIDDKSG (SEQ ID NO:5), IFVIDDK (SEQ ID NO:39), IFVIDDKS (SEQ ID NO:40) or IFVIDDKSG (SEQ ID NO:6), or an analogue or peptidomimetic of any of the foregoing peptide sequences. Within other embodiments, a modulating agent may comprise a cyclic peptide having one of the following sequences: CDDKC (SEQ ID NO:7), CIDDKC (SEQ ID NO:41), CDDKSC (SEQ ID NO:42), CVIDDKC (SEQ ID NO:8), CIDDKSC (SEQ ID NO:9), CVIDDKSC (SEQ ID NO:43), CDDKSGC (SEQ ID NO:44), CIDDKSGC (SEQ ID NO:45), CVIDDKSGC (SEQ ID NO:31), CFVIDDKC(SEQ ID NO:46), CFVIDDKSC (SEQ ID NO:47), CFVIDDKSGC (SEQ ID NO:10), CIFVIDDKC (SEQ ID NO:48), CIFVIDDKSC (SEQ ID NO:49), or CIFVIDDKSGC (SEQ ID NO:11), DDDK(SEQ ID NO:50), DIDDK (SEQ ID NO:51), DVIDDK (SEQ ID NO:52), DFVIDDK (SEQ ID NO:53), DIFVIDDK (SEQ ID NO:54), EDDK(SEQ ID NO:55), EIDDK (SEQ ID NO:56), EVIDDK (SEQ ID NO:57), EFVIDDK (SEQ ID NO:58), EIFVIDDK (SEQ ID NO:59), KDDKD (SEQ ID NO:14), KIDDKD (SEQ ID NO:60), KDDKSD (SEQ ID NO:61), KVIDDKD(SEQ ID NO:62), KIDDKSD (SEQ ID NO:63), KVIDDKSD (SEQ ID NO:64), KDDKSGD (SEQ ID NO:65), KIDDKSGD (SEQ ID NO:26), KVIDDKSGD (SEQ ID NO:66), KFVIDDKD (SEQ ID NO:67), KFVIDDKSD (SEQ ID NO:68), KFVIDDKSGD (SEQ ID NO:69), KIFVIDDKD (SEQ ID NO:70), KIFVIDDKSD (SEQ ID NO:71), KIFVIDDKSGD (SEQ ID NO:72), VIDDK (SEQ ID NO:73), IDDKS (SEQ ID NO:74), VIDDKS (SEQ ID NO:75), DDKSG (SEQ ID NO:76), IDDKSG (SEQ ID NO:24) KDDKE (SEQ ID NO:77), KIDDKE (SEQ ID NO:78), KDDKSE (SEQ ID NO:79), KVIDDKE (SEQ ID NO:80), KIDDKSE (SEQ ID NO:81), KVIDDKSE (SEQ ID NO:82), KDDKSGE (SEQ ID NO:83), KIDDKSGE (SEQ ID NO:84), KVIDDKSGE (SEQ ID NO:85), KFVIDDKE (SEQ ID NO:86), KFVIDDKSE (SEQ ID NO:87), KFVIDDKSGE (SEQ ID NO:88), KIFVIDDKE (SEQ ID NO:89), KIFVIDDKSE (SEQ ID NO:90), or KIFVIDDKSGE (SEQ ID NO:91). In certain embodiments, a modulating agent may be linked to a drug, a detectable marker, a targeting agent and/or a support material. Modulating agents may also, or alternatively, comprise one or more of: (a) a cell adhesion recognition sequence other than an OB-cadherin CAR sequence; and/or (b) an antibody or antigen-binding fragment thereof that specifically binds to a CAR sequence other than an OB-cadherin CAR sequence.
Within further aspects, pharmaceutical compositions are provided, comprising a cell adhesion modulating agent according to claim 1, in combination with a pharmaceutically acceptable carrier. Such compositions may additionally comprise a drug and/or one or more of: (a) a peptide comprising a cell adhesion recognition sequence other than an OB-cadherin CAR sequence; and/or (b) an antibody or antigen-binding fragment thereof that specifically binds to a CAR sequence other than an OB-cadherin CAR sequence.
The present invention further provides, within other aspects, methods for modulating cell adhesion, comprising contacting an OB-cadherin-expressing cell with a cell adhesion modulating agent or a pharmaceutical composition as described above. Such modulating agents and compositions may inhibit or enhance cell adhesion.
Within other aspects, the present invention provides methods for inhibiting adhesion of OB-cadherin expressing cells in a mammal, comprising administering to a mammal a cell adhesion modulating agent as described above, wherein the modulating agent inhibits OB-cadherin-mediated cell adhesion.
The present invention further provides methods for enhancing the delivery of a drug to a tumor in a mammal, comprising administering to a mammal: (a) a modulating agent as described above, wherein the modulating agent inhibits OB-cadherin-mediated cell adhesion; and (b) a drug. The modulating agent may be administered to the tumor or systemically, and may be linked to the drug and/or a targeting agent.
Within other aspects, the present invention provides methods for treating a cancer and/or inhibiting metastasis of a cancer in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent inhibits OB-cadherin-mediated cell adhesion.
The present invention further provides, within other aspects, methods for stimulating blood vessel regression in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent inhibits OB-cadherin-mediated cell adhesion.
In still further aspects, methods are provided for enhancing wound healing in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent enhances OB-cadherin-mediated cell adhesion. Such modulating agents may be linked to a support material.
Within other aspects, the present invention provides methods for enhancing adhesion of foreign tissue implanted within a mammal, comprising contacting a site of implantation of foreign tissue in a mammal with a modulating agent as described above, wherein the modulating agent enhances OB-cadherin-mediated cell adhesion. Such foreign tissue includes skin grafts and organ implants.
In further aspects, methods are provided for increasing vasopermeability in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent inhibits OB-cadherin mediated cell adhesion.
Within other aspects, the present invention provides methods for enhancing drug delivery to the central nervous system of a mammal, comprising administering to a mammal a drug and a modulating agent as described above, wherein the modulating agent inhibits OB-cadherin mediated cell adhesion.
Within further aspects, methods are provided for detecting the presence of OB-cadherin-expressing cells in a sample, comprising: (a) contacting a sample with an antibody or an antigen-binding fragment thereof that binds to an OB-cadherin CAR sequence under conditions and for a time sufficient to allow formation of an antibody-cadherin complex; and (b) detecting the level of antibody-cadherin complex, and therefrom detecting the presence of cadherin expressing cells in a sample.
The present invention further provides kits for detecting the presence of OB-cadherin-expressing cells in a sample, comprising: (a) an antibody or antigen-binding fragment thereof that binds to an OB-cadherin CAR sequence; and (b) a detection reagent.
Within further aspects, the present invention provides methods for identifying a compound capable of modulating OB-cadherin-mediated cell adhesion, comprising: (a) contacting an antibody or antigen-binding fragment that specifically binds to an OB-cadherin CAR sequence with a test compound; and (b) detecting the level of antibody or fragment that binds to the test compound, and therefrom identifying a compound capable of modulating cadherin-mediated cell adhesion.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.